Aminopeptidase derived from bacillus licheniformis, gene encoding the aminopeptidase, expression vector containing the gene, transformant and method for preparation thereof

ABSTRACT

The present invention relates to novel aminopeptidase derived from  Bacillus licheniformis,  a gene encoding the aminopeptidase, an expression vector containing the gene, a cell transformant transfected with the expression vector and a process for preparing a natural type protein using thereof. More particularly, the present invention relates to a gene encoding aminopeptidase which is cloned and manufactured using the recombinant DNA technique, an expression vector containing the gene, a cell transformant transfected with the expression vector and a recombinant aminopeptidase which is necessary to produce recombinant human growth hormone in a natural type protein and can be expressed in a high yield more stably and advantageously, compared with conventional methods for the purification.

RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 national phase application ofPCT/KR02/01280 (WO 03/004635) filed on Jul. 6, 2002, entitled “NovelAminopeptidase Derived from Bacillus Licheniformis, Gene Encoding theAminopeptidase, Expression Vector Containing the Gene, Transformant andMethod for Preparation Thereof” which claims priority to KoreanApplication No. 2001-0040268, filed Jul. 6, 2001 and Korean ApplicationNo. 2002-0030798, filed May 31, 2002, both of which are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to novel aminopeptidase derived fromBacillus licheniformis, a gene encoding the aminopeptidase, anexpression vector containing the gene, a cell transformant transfectedwith the expression vector and a process for preparing a natural typeprotein using thereof. More particularly, the present invention relatesto a gene encoding aminopeptidase which is cloned and manufactured usingthe recombinant DNA technique, an expression vector containing the gene,a cell transformant transfected with the expression vector and arecombinant aminopeptidase which is necessary to produce recombinanthuman growth hormone in a natural type protein and can be expressed in ahigh yield more stably and advantageously, compared with conventionalmethods for the purification.

BACKGROUND ART

Generally, recombinant proteins are made as non-natural types when theyare expressed in a large scale through the gene manipulation inmicrobes. In detail, most of the recombinant proteins having ainitiator, methionine(MET), in the amino terminus, which were treatedinappropriately are produced. The recombinant proteins containingmethionine at the amino terminus might induce immunogenic reactions orbecome being unstable so as not to play an intrinsic role of theprotein, in case of being administered to human or other animals.Therefore, it is very important to develop a new method for preparingsuch a recombinant protein as its natural type protein (See Nature,1987, 326, 315; J. Bacteriol., 1987, 169, 751–757; Bio/Technology, 1990,8, 1036–1040; Appl. Microbiol. Biotechnol., 1991, 36, 211–215).

Human growth hormone is a polypeptide hormone which has 22,125 Da ofmolecular weight, contains a sequence of Phe-Pro-Thr at the aminoterminus and is composed of 191 amino acids secreted from humanpituitary gland and mostly has been applied to treat pituitary dwarfism(See Raben, M. S., J. Clin. Endocr., 1958, 18, 901). Up to now, humangrowth hormone has been extracted and purified from pituitary glands ofthe dead, but is hard to be provided for all the patients due to thelimited supply. Recently, several studies regarding expression andseparation of the human growth hormone from Escherichia coli, yeast andthe like by gene manipulation have been performed. Actually, the humangrowth hormone produced through the DNA technology has been used forclinical applications (in case of Escherichia coli, See Korean PatentPublication No. 89-1244 and No. 87-701; Korean Patent Laid-open No.87-2258 and No. 84-8695; in case of yeast, See Korean Patent PublicationNo. 92-99; Korean Patent Laid-open No. 90-9973 and No. 90-9976).

However, if a human growth hormone is produced by using the recombinantDNA technology described above, one methionine residue, an initiationcodon for protein synthesis is bound additionally at the amino terminusexcept 191 amino acid residues consisting in a natural type human growthhormone and thus a methionyl human growth hormone which is started fromthe sequence of Met-Phe-Pro-Thr of the amino terminus and composed of192 amino acid residues is produced. The methionyl human growth hormonehas the same biological activities as that of the natural type of humangrowth hormone (Moore, J. A., Endocrinology, 1988, 122, 2920–2926) andis not reported yet to provoke side effects by presence of themethionine at the amino terminus. However, antibodies might be generatedon rare occasions due to the methionine and are reported to be producedin a higher ratio than those from the natural type hormone (Lancet,1986, Mar. 29, 697).

Hence, there are several approaches to produce a natural type hormonewhich do not contain the methionine. Concretely, the human growthhormone is produced by one method in which the amino terminus is fusedwith the carboxy terminus of another protein; and then digested by usinga specific protease (See PCT International Application WO 89/12678;European Patent Application EP 20209; European Patent EP 321940); and byanother method which comprises (1) expressing growth hormone withincells; (2) digesting methionine while secreted out of host cells; and(3) obtaining a natural type human growth hormone from culture media(See European Patent EP 008832; U.S. Pat. No. 4,755,465; Japanese PatentJP 01273591; European Patent Application EP 306673; Korean PatentApplication No. 92-10932). But, there are some disadvantages in thesemethods illustrated above. Precisely, it is required of complicatingconstruction a new expression vectors and steps of transforming hostcells and coincidently to optimize the expression conditions with extratrials.

On the other hand, in case that exogenous protein produced by usingrecombinant DNA technologies include an extra amino acid at the aminoterminus, the aminopeptidase can be exploited to remove the extra aminoacid and as a result, the exogenous protein having the same structurewith the natural type protein can be yielded easily. Concretely,specific aminopeptidase which cuts selectively only methionine residuepresent at the amino terminus of the methionyl human growth hormonepurified through conventional methods can be used in order to produce anatural type human growth hormone (See PCT International Application WO86/04609 and WO 86/204527 A1).

Presently, various kinds of aminopeptidases have been demonstrated toprepare a natural type human growth hormone. In detail, theaminopeptidase purified from Aeromonas proteolytica (See PCTInternational Application WO 86/01229; European Patent EP 0489711, A3,BTG company; Prescott and Wilks, Method in Enzymology, 1976, 44,530–543), the aminopeptidase purified from porcine kidney (See PCTInternational Application WO 86/204527 A1; Bio/Technology, 1987, 5,824–827, Takeda company), the dipeptidyl aminopeptidase purified fromDictyostelium discoidium (See European Patent EP 557076; U.S. Pat. No.5,126,249, A1, Eli Lilly company), and the aminopeptidase fromStreptomyces thermonitripican were reported.

In order to attain the above object, the aminopeptidase should not workon the amino acid sequence of a natural type protein although it removesunnecessary amino acid residues present at the amino terminus of therecombinant protein. Namely, when the original protein has a amino acidsequence starting from X-Y-Z at the amino terminus and the recombinantprotein has the amino acid sequence of Met-X-Y-Z-containing anadditional methionine at the amino terminus, the aminopeptidase shouldcut only the methionine residue and not work onto other amino acids(X-Y-Z-) afterward so as to produce the recombinant protein having thesame amino acid sequence as that of the natural type protein. Therefore,the aminopeptidase satisfying such an object should be compatible in itssubstrate specificity depending upon the target protein for industrialapplications. Although the enzyme has such a characteristic, it isadvantageous to have higher intrinsic activities itself.

Presently, more than several tens of aminopeptidases have been extractedfrom microbes and the like and disclosed. In common, most enzymes needmetal ions such as calcium, zinc or the like in order to be activated.These aminopeptidases have various properties in view of molecularweight, essential metal ions, optimal condition of the reaction,substrate specificity and so on according to microbes, although theyhave common activities in view of the digestion in amino acid residuesat the amino terminus of the protein (See FEMS Microbiol. Rev., 1996,18, 319–344). Such an aminopeptidase is classified to an exopeptidaseand has a property to isolate a amino acid from the amino terminus ofsubstrate protein in orders.

Precisely, these aminopeptidases have been reported and separated,especially from Bacillus sp., for example Bacillus subtilis (See Arch.Biochem. Biophys., 1979, 197, 63–77; Arch. Biochem. Biophys., 202,540–545, 1980; J. Biochem., 1994, 107, 603–607; Japanese Patent JP03285684, Diacel-Chem company), Bacillus stearothermophilus (See Meth.Enzymol., 1970, 19, 544–552; Biochem. Biophys. Acta, 1976, 438, 221–220;European Patent EP 101653, Unitika company), Bacillus thuringensis(Biokhimiya, 1984, 49, 1899–1907), Bacillus licheniformis (Arch.Biochem. Biophys., 1978, 186, 383–391; Mikrobiol. Zh., 1989, 51, 49–52)and so on.

In the references mentioned above, the aminopeptidases are purified andanalyzed for their enzymatic properties by using leucine-p-nitroanilide,and several dipeptides or the like as a substrate. However,oligopeptides starting from the sequence of Met-X-Pro at the aminotermini or proteins starting from Met-X-Pro at the amino termini are notused as a substrate in order to measure enzymatic activities. Therefore,it is not verified yet that these aminopeptidases might be applied tothe methionine removal in recombinant proteins containing Met-X-Prosequence at their amino termini.

Furthermore, the aminopeptidases derived from Bacillus licheniformishave been illustrated as follows. Rodriguez et al. have purified theaminopeptidase derived from Bacillus licheniformis ATCC 12759 andexamined its enzymatic property. Also, the inventors of the presentinvention have disclosed the method for preparing a natural type humangrowth hormone using the aminopeptidase derived from Bacilluslicheniformis, the characterization of the purified aminopeptidase inits enzymatic properties and its amino acid sequence at the aminoterminus (Korean Patent Laid-open No. 1998-071239). In this invention,for producing a natural type protein in a large scale throughrecombinant DNA technologies, the inventors of the present inventionhave reported that the aminopeptidase could remove only methionineresidue in synthetic substrates, oligopeptides, proteins and the likeand recognize the specific amino acid sequence of Met-X-Pro(hereinafter, X denotes any amino acid regardless of its kind) and thusbe suitable for the production of the natural type human growth hormone(Korean Patent Laid-open No. 1998-071239). These conventional methodshave elucidated that the aminopeptidase can be produced from Bacilluslicheniformis and exploited to produce a natural type recombinantprotein. Besides, they have provided only the information about thepartial amino acid sequence of the amino terminus and the total gene ofaminopeptidase has not determined yet.

Hence, the inventors of the present invention have tried to obtain novelaminopeptidase for producing a natural type recombinant protein.Concretely, we have cloned a gene of aminopeptidase derived fromBacillus licheniformis, determined its nucleotide sequence and aminoacid sequence and expressed the cloned aminopeptidase gene inrecombinant bacterial strains. Then, the recombinant protein wasidentified to have aminopeptidase activities, to be useful for useeasily other enzymatic reaction as well as the preparation of naturaltype proteins. Consequently, the present invention has been completedsuccessfully.

DISCLOSURE OF INVENTION

The object of the present invention is to provide novel aminopeptidasederived from Bacillus licheniformis, a gene encoding the aminopeptidase,an expression vector containing the gene, a cell transformanttransfected with the expression vector and a process for preparing anatural type protein using thereof, which can be used to manufacture arecombinant protein as well as applied to other enzymatic reactionsusefully.

In order to accomplish the object described above, the present inventionprovides an aminopeptidase derived from Bacillus licheniformis.

Precisely, the aminopeptidase has an amino acid sequence selected fromthe group comprising the amino acid sequence having the full length ofSEQ. ID NO: 1 or an amino acid sequence selected from a sequence whichcomprises the amino acid of SEQ. ID NO: 1 having a deletion of one ormore amino acids.

Preferably, the aminopeptidase has an amino acid sequence selected fromthe group comprising the amino acid sequence of SEQ ID NO:1 having adeletion of one or more amino acids at the amino terminus, carboxyterminus or both termini.

More preferably, the aminopeptidase is selected from a group ofaminopeptidases having a deletion at the amino terminus of SEQ ID NO:1wherein said amino acid sequence is selected from the group consistingof: an amino acid sequence having a deletion of amino acids 1 through 30of SEQ. ID NO: 1; an amino acid sequence having a deletion of aminoacids 1 through 39 of SEQ. ID NO: 1; an amino acid sequence having adeletion of amino acids 1 through 40 of SEQ. ID NO: 1; an amino acidsequence having a deletion of amino acids 1 through 41 of SEQ. ID NO: 1;and an amino acid sequence having a deletion of amino acids 1 through 42of SEQ. ID NO: 1. In addition the aminopeptidase is selected from thegroup consisting of the above referenced sequences having a furtherdeletion of amino acids 444 through 449 at the carboxy terminus of SEQID NO:1.

In addition, the aminopeptidase has the amino acid sequence of SEQ. IDNO: 1 having a deletion of amino acids 444 through 449 at the carboxyterminus.

In addition, the present invention provides a gene encoding theaminopeptidase derived from Bacillus licheniformis.

Precisely, the gene encoding the aminopeptidase has a nucleotidesequence that encodes an aminopeptidase selected from the groupconsisting of SEQ ID NO:1, or a fragment thereof wherein said fragmentis selected from the group consisting of: an amino acid sequence havinga deletion of amino acids 1 through 30 of SEQ. ID NO: 1; an amino acidsequence having a deletion of amino acids 1 through 39 of SEQ. ID NO: 1;an amino acid sequence having a deletion of amino acids 1 through 40 ofSEQ. ID NO: 1; and an amino acid sequence having a deletion of aminoacids 1 through 41 of SEQ. ID NO: 1; an amino acid sequence having adeletion of amino acids 1 through 30 and 444 through 449 of SEQ. ID NO:1; an amino acid sequence having a deletion of amino acids 1 through 39and 444 through 449 of SEQ. ID NO: 1; an amino acid sequence having adeletion of amino acids 1 through 40 and 444 through 449 of SEQ. ID NO:1; and an amino acid sequence having a deletion of amino acids 1 through41 and 444 through 449 of SEQ. ID NO: 1 and an amino acid sequencehaving a deletion of amino acids 444 through 449 of SEQ ID NO:1.

In addition, the present invention provides an expression vector pLAP132which contains the gene encoding the aminopeptidase with the nucleotidesequence in the full length of SEQ. ID NO: 2.

Besides, the present invention provides an Escherichia coli transformantXLOLR/LAP132 which is transfected with the expression vector pLAP132(accession number: KCTC 1000 BP).

Furthermore, the present invention provides a process for preparing apeptide or protein having a native amino-terminus lacking a methionineamino acid which comprises the following steps: (1) purifying arecombinant protein containing a methionine-X-proline sequence at theamino terminus; (2) adding an aminopeptidase to said purifiedrecombinant protein; and (3) digesting said mixture thereby obtainingsaid protein or peptide having a native amino-terminus lacking amethionine amino acid.

At this moment, X of the Met-X-Pro can be any kind of amino acid.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which;

FIG. 1 depicts the determination of amino acid sequences in peptidefragments of the aminopeptidase obtained after treating trypsin.

FIG. 2 depicts the analysis of the aminopeptidase derived from Bacilluslicheniformis and expressed from Escherichia coli transformant byperforming SDS-polyacrylamide gel electrophoresis.

FIG. 3 depicts the analysis of the aminopeptidase derived from Bacilluslicheniformis and expressed from Bacillus subtilis transformant byperforming SDS-polyacrylamide gel electrophoresis.

FIG. 4 depicts the examination of enzymatic activities in theaminopeptidase derived from Bacillus licheniformis and expressed fromBacillus subtilis transformant schematically.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be illustrated more clearly asfollows.

The aminopeptidase of the present invention is identified to haveenzymatic activities in a polypeptide state before signal peptide is notdeleted (the sequence composed of 1st amino acid through 30th amino acidin SEQ. ID NO: 1) and after signal peptide is deleted. Although someamino acids at the amino terminus and at the carboxy terminus are cut inaddition to the deletion of signal peptide, the enzymatic activities aremaintained. Therefore, the aminopeptidase containing the amino acidsequence of SEQ. ID NO: 1 as well as the aminopeptidases deletedpartially at the amino terminus or at the carboxy terminus from theamino acid sequence of SEQ. ID NO: 1 can be within the scope of thepresent invention.

Preferably, the aminopeptidase of the present invention is selected froma group of aminopeptidases having a deletion at the amino terminus ofSEQ ID NO:1 wherein said amino acid sequence is selected from the groupconsisting of: an amino acid sequence having a deletion of amino acids 1through 30 of SEQ. ID NO: 1; an amino acid sequence having a deletion ofamino acids 1 through 39 of SEQ. ID NO: 1; an amino acid sequence havinga deletion of amino acids 1 through 40 of SEQ. ID NO: 1; an amino acidsequence having a deletion of amino acids 1 through 41 of SEQ. ID NO: 1;and an amino acid sequence having a deletion of amino acids 1 through 42of SEQ. ID NO: 1. More preferably, the aminopeptidase of the presentinvention has a deletion of amino acids 1 through 30 of SEQ. ID NO: 1.

Preferably, the aminopeptidase of the present invention has the aminoacid sequence of SEQ. ID NO: 1 having a deletion of amino acids 444through 449 at the carboxy terminus.

More preferably, the aminopeptidase has the amino acid sequence of SEQID NO: 1 having a deletion of amino acids 1 through 42 and 444 through449.

The genes of the present invention encoding the aminopeptidase derivedfrom Bacillus licheniformis can include the gene encoding amino acidsequence of SEQ. ID NO: 1, the gene encoding all of the deleted form ofaminopeptidase mentioned above and the gene of SEQ. ID NO: 2.

In order to investigate functions and enzymatic activities of theaminopeptidase, the following procedure is accomplished by using theprotein containing the amino acid sequence of SEQ. ID NO: 1, its genesand its polypeptide in a deleted form respectively.

In order to elucidate polypeptides having enzymatic activities of theaminopeptidase derived from Bacillus licheniformis, a gene encoding theaminopeptidase is cloned from the chromosomal DNA of Bacilluslicheniformis. Concretely, for cloning genes, polypeptides having theenzymatic activities of the aminopeptidase derived from Bacilluslicheniformis are purified, digested with trypsin so as to collect anumber of peptide fragments and then determined the amino acidsequences. The information of the amino acid sequences is exploited tosynthesize oligonucleotides for use of primers and then DNA fragmentscorresponding to a part of the aminopeptidase gene are amplified.Afterward, by utilizing these DNA fragments for probes, theaminopeptidase gene can be found from the chromosomal library derivedfrom Bacillus licheniformis.

The genes of aminopeptidase observed above are examined to determine thenucleotide sequences with the DNA sequence analyzer as shown in SEQ. IDNO: 2, and the deduced DNA sequence from the genetic information of SEQ.ID NO: 1 is identified to have exactly the same amino acid sequence ofthe aminopeptidase purified from natural host cells. As a result, theaminopeptidase polypeptide obtained according to the present inventionis screened by using data base for genetic informations and this gene isidentified a novel DNA sequence that have never reported and have aenzymatic activities of the aminopeptidase when it is expressed inrecombinant microbes.

Then, the matured aminopeptidase is also detected to be digestedpartially. In order to investigate the composition of the aminopeptidaseat the carboxy terminus, the aminopeptidase purified from therecombinant bacterial transformant and the aminopeptidase purified fromnatural host cells, Bacillus licheniformis, are examined to determinemolecular weights with mass spectrometry. Consequently, in both cases 6amino acid residues are verified to be cut from the carboxy terminus.

The aminopeptidase according to the present invention is confirmed tobecome mature that the aminopeptidase were expressed in the cell andthen, the signal peptide was deleted at the amino terminus during theextracellular secretion. Besides, the matured aminopeptidase is alsodigested partially.

Hence, the aminopeptidase polypeptide according to the present inventionmaintains enzymatic activities with presence of the signal peptide andeven partially being cut at the amino terminus or at the carboxyterminus with absence of the signal peptide.

EXAMPLES

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Cloning of aminopeptidase gene derived from Bacilluslicheniformis

(1-1) Partial determination of amino acid sequence in aminopeptidasepurified from Bacillus licheniformis

The present inventors have performed the procedure as follows in orderto purify aminopeptidase protein from Bacillus licheniformis.

Sterilized media containing tryptone 10 g, yeast extract 5 g, NaCl 10 gper 1 L were prepared in flasks and Bacillus licheniformis KCTC 3058strain was inoculated so as to be cultivated for seed-culture at 40° C.,120 rpm for about 16 hours. The seed culture broth obtained above wasinoculated again to new media and cultivated at 40° C., 200–400 rpmagitation, more than 30 of dissolved oxygen. Then, the concentrations ofglucose were measured with an hour interval and the cultivation wascompleted when the activity of aminopeptidase reached more than 35 U/ml.In order to cultivate cells, culture broth containing peptone 2 kg,yeast extract 6 kg, potassium phosphate 4 kg, NaCl 1 kg, SAG, MgSO₄·7H₂O15 g, FeSO₄·7H₂O 1.53 g, ZnSO₄·7H₂O 1.53 g, MnSO₄ 1.53 g, and glucose 10kg per distilled water 200 L in 400 L fermentor was prepared in asterilized state. After the cultivation was completed, continuouscentrifuge was utilized to remove cell debris and recovered thesupernatants. The supernatant obtained above was concentrated with aconcentrator and then ZnSO₄ was added to reach about 0.3 mM. Through 3steps of column chromatography, aminopeptidase was purified from theconcentrated solution. As a chromatography, SP-Sepharose FF, SephacrylS-200, and DEAE-Sepharose FF were utilized in turns. The aminopeptidasepurified by using the above procedure was identified to have more than95% of purity when analyzed with the reverse phase high pressurechromatography.

The resulted aminopeptidase was precipitated by using 10%trichloroacetic acid (TCA), washed with acetone and dried. The driedprotein precipitate was dissolved with 8 M urea and 0.4 M ammoniumbicarbonate and treated with dithiothreitol(DTT) and iodoacetamide inturns so that disulfide bonds in the aminopeptidase were cleaved. Thetreated sample was dissolved again in distilled water, added about 0.05mg of trypsin per 2 mg of aminopeptidase and treated at 37° C. for about8 hours. The sample treated with trypsin was injected into the reversephase high pressure chromatography (RP-HPLC) and collected to fractionsof major peptide peaks. In the reverse phase chromatography, Vydac C18reverse phase column was adopted and a linear concentration gradientusing solvent A (highly purified water containing 0.05% trifluoroaceticacid(TFA)) and solvent B (highly purified water containing 0.05% TFA and80% acetonitrile) was applied. At this moment, the analysis wasperformed in 0.5 ml/min of flow velocity and chromatogram was obtainedby UV absorbance at 214 nm. Through this procedure, more than 10 peakswere obtained. The resulted peptide sample was analyzed with a massspectrometry respectively and peptides composed of more than 15 aminoacids were selected. The selected sample was determined the amino acidsequence with the amino acid sequence analyzer. FIG. 1 depicted theamino acid sequence of the selected peptide by using 1 characterdenoting an amino acid. Each peptide was numbered in orders arbitrarilyin accordance with elution time through reverse phase chromatography andthe arrow part depicted in FIG. 1 was used to give information forsynthesizing the primers of oligonucleotides. The sample denoted as T4among these peptide samples were deduced to contain 2 different peptidescoincidently since 2 amino acids appeared at the same time in each cycleby performing the sequence analysis of amino acids. Besides, among thesepeptide samples, the same sequence of amino acids is contained inbetween T6 and T9, T11 and T13 and T16 and T17. Precisely, T4 describedin SEQ. ID NO: 4 and 5, T6 in SEQ. ID NO: 6, T7 in SEQ. ID NO: 7, T9 inSEQ. ID NO: 8, T11 in SEQ. ID NO: 9, T13 in SEQ. ID NO: 10, T16 in SEQ.ID NO: 11, T17 in SEQ. ID NO: 12 respectively, which is disclosed inSequence List independently.

(1-2) Cloning of aminopeptidase gene and determination of nucleotidesequence

By using the peptide information elucidated in Example 1(1-1),oligonucleotide primers for PCR of aminopeptidase genes weresynthesized. Precisely, 5′- upstream primer(LAP-5) described in SEQ. IDNO: 13 and 3′-downstream primer(LAP-3) described in of SEQ. ID NO: 14were utilized, which were manufactured by using the amino acid sequencesdescribed in SEQ. ID NO: 15 and SEQ. ID NO: 16 among amino acidsequences determined in Example 1 (1-1). PCR was performed 32 timesrepeatedly with the DNA thermal cycler; denaturation at 94° C. for 30seconds, annealing at 40° C. for 45 seconds, and extension at 72° C. for1 minute and chromosomal DNA of Bacillus licheniformis was used as atemplate. As a result, DNA fragment with about 390 bp size was obtained.

In the meantime, genomic DNA of Bacillus licheniformis was made byexploiting Murray and Thompson's method and digested partially with therestriction enzyme Sau3A and separated it through 0.8% agarose gel thenisolated the DNA fragments corresponding to 2˜3 kb. The DNA fragment wasligated into λ ZAP expression vector (Stratagene, LaJolla, USA) digestedwith BamHI and added to packaging extract. Then, the packaging mixturewas transferred to E. coli XL-1 Blue MRF′. This library filter preparedabove was screened by using ³²P-labelled PCR fragment and detected forclones containing aminopeptidase gene. As a result, the selected clonewas verified to include DNA insert with about 2.6 kb size and named it“LAP 132” clone.

The nucleotide sequence of LAP 132 was analyzed and the result wasdescribed in SEQ. ID NO: 2. In detail, it was composed of ATG initiationcodon starting at 192 nucleotide and TAA termination codon at 1,539 andcontained 1,347 bp ORF (open reading frame) as shown in SEQ. ID NO: 2and in SEQ. ID NO: 3. The nucleotide was identified to encode 449 aminoacids. The amino acid sequence encoded from the nucleotide sequenceinformation elucidated above, were identical completely with the resultdetermined by the amino acid sequence analysis of peptide fragmentsderived from aminopeptidase purified above. The domain of amino terminusof the aminopeptidase according to the present invention containedhydrophobic amino acid residues contiguous to cationic amino acidresidues, which was similar to the signal sequence needed for thesecretion. Thus, the aminopeptidase was deduced to be digested inbetween 30th alanine and 31st alanine when secreted. But, in KoreanPatent Laid-open No. 1998-071239, the aminopeptidase was disclosed thatamino terminus mainly starts from amino acid sequences described in SEQ.ID NO: 17 and heterogeneous type forms of aminopeptidases of which theircleaved sites are somewhat different with the present invention. Thisdifference was deduced to be provoked by digesting with the otherextracellular protease after the aminopeptidase was secreted fromoriginal strains. The aminopeptidase according to the present inventionwas compared in the amino acid sequence with other aminopeptidaserecorded in Genebank by using BLAST searching. As a result, it was shownto have 62% homology with the aminopeptidase derived from Bacillussubtilis, and 58% homology with aminopeptidase derived from Bacillushalodurans. The aminopeptidase according to the present inventionidentified as a new aminopeptidase not reported previously.

The inventors of the present invention have transformed theaminopeptidase gene, “LAP 132” derived from Bacillus licheniformis, intoE. coli XLOLR strain and have named with “E. coli XLOLR/LAP 132”. Theyhave deposited with International Deposit Organization, the KoreanCollection for Type Cultures (KCTC) of the Korean Research Institute ofBioscience and Biotechnology (KRIBB), Republic of Korea on Apr. 26,2001, and identified as accession number, KCTC 1000 BP.

Example 2 Gene expression of aminopeptidase in Escherichia colitransformant

The aminopeptidase gene cloned according to the present invention wassubcloned into the expression vector pBK-CMV (Stratagene, USA) and namedthe expression vector “pLAP32” so as to be used as a source ofaminopeptidase gene. Then, the PCR fragment with about 1.2 kb sizecontaining a region encoding aminopeptidase was subcloned into thevector pET11a (Stratagene, USA) and named the expression vectorpETLAP45. The expression vector was transformed into E. coli BL21(DE3)and exploited it to express a fused protein attaching His-tag peptide.The expression of aminopeptidase in the expression system describedabove was verified by performing SDS-PAGE. As illustrated in FIG. 2, themolecular weight of aminopeptidase was examined to reach about 45 kDaonto SDS-PAGE and identical to that deduced from its gene. In FIG. 2,lane M depicted a standard marker of molecular weights; lane 1, E. colitransformed into the expression vector pET11a (not inducing theexpression); lane 2, E. coli transformed into the expression vectorpET11a (inducing the activation of T7 promoter); lane 3, E. colitransformed into the expression vector pETLAP45 (not included theexpression); and lane 4, E. coli transformed into the expression vectorpETLAP45 (inducing the activation of T7 promoter). The arrow depicted inFIG. 2 denoted the aminopeptidase.

The inventors of the present invention have tried to detect enzymaticactivities of aminopeptidase secreted from E. coli transformant afterexpression the gene. Above all, when the E. coli transformant wassonicated and analyzed, the enzymatic activities of the aminopeptidasefrom the E. coli transformant were found in soluble fractions. As aresult, the aminopeptidase according to the present invention wasidentified to have the same size with that deduced from the gene and toshow the enzymatic activities.

Example 3 Gene expression of aminopeptidase in Bacillus Subtilistransformant, sequence analysis of the purified aminopeptidase at theamino terminus and determination of its molecular weight

(3-1) Gene expression of aminopeptidase in Bacillus licheniformistransformant

In the present invention, DNA fragment digested with HindIII-SacI,containing a region encoding aminopeptidase as well as promoter,3′-untranslated region, having about 2.6 kb size and was subcloned intothe Bacillus vector pRB373 and the resulted expression vector was namedpRB373-LAP. The expression vector was transformed into Bacillussubtilis, and cultivated and then the cultured broth was analyzed byusing SDS-PAGE. The aminopeptidase was observed to be secreted from thecultured solution and to have about 45 kDa of molecular weight, whichwas compatible with that from Bacillus licheniformis (See FIG. 3). InFIG. 3, lane M depicted a standard marker of molecular weight; lane 1,Bacillus subtilis transformed into the expression vector pRB373; lane 2,Bacillus subtilis transformed into the expression vector pRB373-LAP; andlane 3, aminopeptidase purified from Bacillus licheniformis. The arrowdepicted in FIG. 3 denoted the aminopeptidase.

(3-2) Sequence analysis of the aminopeptidase polypeptide at the aminoterminus expressed from Bacillus subtilis transformant

In order to purify the aminopeptidase, recombinant Bacillus subtilistransformant containing the gene according to the present invention wascultivated and the cultured broth was separated by performingSP-Sepharose chromatography. The amino acid sequence at the aminoterminus of the purified aminopeptidase was determined by the amino acidanalyzer. As a result, it was verified that the sequence ofaminopeptidase at the amino terminus was heterogeneous as found in theaminopeptidase derived from natural host cells and initiated with SEQ.ID NO: 17 mostly among those with SEQ. ID NO: 18. And the aminopeptidaseinitiated with Asn, Val and Glu also existed.

(3-3) Determination of molecular weight in aminopeptidase polypeptideexpressed from recombinant Bacillus subtilis transformant

The recombinant Bacillus subtilis transformant containing theaminopeptidase gene was cultivated and the cultured broth was exploitedto purify the aminopeptidase by using SP-Sepharose chromatography. Themolecular weight of the aminopeptidase was examined with massspectrometry and as a result, substances corresponding to 42,965 Da ofmolecular weight, 43,241 Da and 43,468 Da appeared. This result wascompared with that obtained from the sequence analysis of amino acids inExample 3(3-2) and additional 6 amino acids was deduced to be removed atthe carboxy terminus. That is to say, the polypeptide described in SEQ.ID NO: 1, the amino terminus started from SEQ. ID NO: 17 and the carboxyterminus ended to SEQ. ID NO: 19 had a theoretical molecular weightcorresponding to about 43,241 Da. Then, another polypeptide added in 2amino acids from the above had a molecular weight corresponding to43,468 Da and the other polypeptide deleted in 2 amino acids had amolecular weight corresponding to 42,965 Da, which was identical to theresults obtained from mass spectrometry exactly.

In the meantime, the aminopeptidase purified from a natural host cell,Bacillus licheniformis was examined by using mass spectrometry throughthe same procedure of Example 1 (1-1) and the same result with that ofrecombinant transformant was obtained.

Example 4 Activity measurement of aminopeptidase and its deleted formsexpressed in recombinant E. coli transformant and Bacillus subtilistransformant

The inventors of the present invention measured the aminopeptidaseactivity using cell lysate solution sonicated in case of recombinant E.coli transformant and using cultured solution obtained in Example 3(3-1) in case of recombinant Bacillus subtilis transformant.

Concretely, Pflleiderer's method was utilized in order to estimateenzymatic activities of the aminopeptidase produced in Example 2 andExample 3 (Pflleiderer, Meth. Enzymol., 1970, 19, 514–521). 50 μl ofcultured solution was added into the mixture of 1 M Tris (pH 8.5)(950μl), and 0.1 M leucine-p-nitroanilide(20 μl) in DMSO, reacted for 3minutes at 60° C., added 100 μl of 70% acetic acid, ended the reactionand detected the absorbance at 405 nm.

Consequently, as shown in FIG. 4, there was a precise difference inbetween Bacillus subtilis strain transformed with the aminopeptidasegene and Bacillus subtilis strain containing the expression vector. Inaddition, the aminopeptidase purified from recombinant Bacillus subtiliswas composed conincidently of deleted forms at the amino terminus or atthe carboxy terminus as demonstrated in Example 3 (3-2) and (3-3) andthe samples were also identified to have enzymatic activities. In FIG.4, □ depicted pRB373-LAP, Bacillus subtilis transformed with theexpression vector containing the aminopeptidase gene; Δ depicted LG,Bacillus subtilis cultivated in LB culture broth; and ♦ depicted pRB373,Bacillus subtilis transformed with the expression vector not containingan aminopeptidase gene.

INDUSTRIAL APPLICABILITY

As demonstrated clearly and confirmed above, according to the presentinvention the gene encoding the aminopeptidase derived from Bacilluslicheniformis is cloned and expressed by using recombinant bacterialtransformant and is identified to be novel gene not reported previously.The aminopeptidase purified and elucidated according to the presentinvention can be exploited usefully to manufacture natural typerecombinant proteins as well as applied to other enzymatic reactionswidely.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1. An isolated aminopeptidase having the amino acid sequence set forthin SEQ ID NO:1.
 2. An isolated aminopeptidase having an amino acidsequence selected from the group consisting of, the sequence of aminoacids from position 31 through position 449 of SEQ ID NO:1; the sequenceof amino acids from position 40 through position 449 of SEQ ID NO:1; thesequence of amino acids from position 41 through position 449 of SEQ IDNO:1; and the sequence of amino acids from position 42 through position449 of SEQ ID NO:1.
 3. An isolated aminopeptidase consisting of thesequence of amino acids from position 1 through position 443 of SEQ IDNO:1.
 4. An isolated aminopeptidase having an amino acid sequenceselected from the group consisting of, the sequence of amino acids fromposition 31 through position 443 of SEQ ID NO:1; the sequence of aminoacids from position 40 through position 443 of SEQ ID NO:1; the sequenceof amino acids from position 41 through position 443 of SEQ ID NO:1; andthe sequence of amino acids from position 42 through position 443 of SEQID NO:1.
 5. An isolated nucleic acid sequence encoding an aminopeptidaseaccording to claim 1, 2, 3, or
 4. 6. The expression vector pLAP132 whichcomprises the nucleotide sequence set forth in SEQ ID NO:2.
 7. AnEscherichia coli transformant XLOR/LAP132 transformed with theexpression vector pLAP132 and having the accession number KCTC 1000 BP.8. A process for preparing a peptide or a recombinant protein lacking amethionine present at its native amino-terminus that comprises thefollowing steps: (1) purifying the peptide or recombinant proteincontaining a methionine-x-proline sequence at the amino terminus whereinthe x of said methionine-x-proline sequence is any amino acid; (2)contacting said purified peptide or recombinant protein with anaminopeptidase of any of claim 1, 2, 3, or 4 under conditions suitablefor cleavage of the amino-terminal methionine from the recombinantprotein; and (3) thereby obtaining said peptide or recombinant proteinhaving an amino-terminus lacking a methionine.